Methods and compositions for the identification, characterization and inhibition of farnesyltransferase

Abstract

Disclosed are methods and compositions for the identification, characterization and inhibition of mammalian protein farnesyltransferases, enzymes involved in the farnesylation of various cellular proteins, including cancer related ras proteins such as p21ras. One protein farnesyltransferase which is disclosed herein exhibits a molecular weight of between about 70,000 and about 100,000 upon gel exclusion chromatography. Also disclosed are methods and compositions for the preparation of farnesyltransferase by recombinant means, following the molecular cloning and co-expression of its two subunits, for assay and purification of the enzyme, as well as procedures for using the purified enzyme in screening protocols for the identification of possible anticancer agents which inhibit the enzyme and thereby prevent expression of proteins such as p21ras. Also disclosed is a families of compounds which act either as false substrates for the enzyme or as pure inhibitors and can therefore be employed for inhibition of the enzyme. The most potent inhibitors are ones in which phenylalanine occurs at the third position of a tetrapeptide whose amino terminus is cysteine.

Description

1. Field of the InventionThis invention relates generally to improved peptide-based inhibitors of farnesyltransferase, the enzyme responsible for the farnesylation of p21.sup.ras protein, and more particularly relates to peptide-based "pure" inhibitors having improved characteristics and structures. Improvements are based on the inventors' discovery of structural characteristics that ensure that a peptide inhibitor will exhibit "pure" inhibitor characteristics, which provide important guidelines for inhibitor design that will allow cellular uptake while preserving inhibitory capabilitites.2. Description of the Related ArtIn recent years, some progress has been made in the elucidation of cellular events lending to the development or progression of various types of cancers. A great amount of research has centered on identifying genes which are altered or mutated in cancer relative to normal cells. In fact, genetic research has led to the identification of a variety of gene families in...

AI Technical Summary

Benefits of technology

As outlined above, the inventors have discovered that the farnesyltransferase enzyme is capable of binding to an affinity chromatography medium comprised of the peptide TKCVIM, coupled to a suitable matrix. This feature of the farnesyltransferase enzyme was discovered by the present inventors in developing techniques for its isolation. Surprisingly, it has been found that the coupling of a peptide such as one which includes CVIM, as does TKCVIM, to a suitable chromatography matrix allows for the purification of the protein to a significant degree, presumably through interaction and binding of the enzyme to the peptidal sequence. A basis for this interaction could be posited as due to the apparent presence of a farnesyl acceptor moiety within this peptide.

Problems solved by technology

There will, of course, be conditions, such as when the pH is below 6.0, wherein the farnesyltransferase enzyme will not bind effectively to such a matrix.

Interestingly, it has been found that slight changes in the sequence of the acceptor site can result in loss of inhibitory activity.

Of course, charged species cross cellular membranes only with some difficulty, if at all.

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